Selection and characterization of human scFvs targeting the SARS-CoV-2 nucleocapsid protein isolated from antibody libraries of COVID-19 patients

In 2019, the novel SARS-CoV-2 coronavirus emerged in China, causing the pneumonia named COVID-19. At the beginning, all research efforts were focused on the spike (S) glycoprotein. However, it became evident that the nucleocapsid (N) protein is pivotal in viral replication, genome packaging and evasion of the immune system, is highly immunogenic, which makes it another compelling target for antibody development alongside the spike protein. This study focused on the construction of single chain fragments variable (scFvs) libraries from SARS-CoV-2-infected patients to establish a valuable, immortalized and extensive antibodies source. We used the Intracellular Antibody Capture Technology to select a panel of scFvs against the SARS-CoV-2 N protein. The whole panel of scFv was expressed and characterized both as intrabodies and recombinant proteins. ScFvs were then divided into 2 subgroups: those that exhibited high binding activity to N protein when expressed in yeast or in mammalian cells as intrabodies, and those purified as recombinant proteins, displaying affinity for recombinant N protein in the nanomolar range. This panel of scFvs against the N protein represents a novel platform for research and potential diagnostic applications.


Dot Blot Assay
Dot Blot Assay was carried out by spotting different concentrations of a recombinant SARS-CoV-2 N protein (RayBiotech) (1 µg, 0.5 µg, 0.25 µg, 0.125 µg, and 0.0625 µg.) in the fixed volume of 1 μL on a nitrocellulose membrane (GE Healthcare).The same volume of proNGF at the concentration of 1.4 µg/μL was spotted as negative control.
The membrane was set in TBS for 3 minutes and incubated for 2 hours and half in agitation with blocking solution (10% non-fat dry milk in 0.05% TBS-Tween (TBST)).The membrane was then incubated overnight at 4°C in 5% blocking solution containing 5 µg/mL of scFv.
After removal of the scFv and washing with 0.05% TBST, the membrane was incubated with a mouse MAb anti-V5 (Invitrogen) at the concentration 1:1000 for 2 hours at room temperature (RT) and then with the secondary antibody, an anti-mouse IgG HRP-conjugated (Peroxidase AffiniPure Goat Anti-Mouse IgG (H+L) Jackson ImmunoResearch) at the concentration 1:7000, for further 2 hours at RT. Spots on the membrane were detected by using ECL (GE healthcare) and an Invitrogen™ iBright™ Imaging System.scFvs cloned in the pEF1α-IRES-ZsGreen1 plasmid were expressed in Vero-TMPRSS2 cells.The scFvs-HA tagged (28 KDa) or an unrelated scFv (#58F), were detected using an anti HA antibody.
Untransfected wild type cells (Wt) and cells transfected with the empty plasmid (Φ) cell extracts were loaded as negative control (upper panel); actin was used as loading control (lower panel).C) Vero-TMPRSS-2 cells were first transfected with the anti-N scFv (cloned in the pEF1α-IRES-ZsGreen1 plasmid) and 48 h post transfection, wild-type cells and cells expressing scFvs-HA were infected with the SARS-CoV-2 B.1 strain at MOI 0.1.The supernatants from the infected cells were harvested 48h post infection.The viral titer was expressed as threshold cycle (Ct) of viral RNA polymerase (RdRp) obtained by real time PCR, in the right graph the viral titer was expressed as logarithm of tissue culture infectious dose 50 (TCID50), i.e. the dilution of a virus required to infect 50% of a cell culture.No statistically significant reduction in virus titer was detected in any sample compared to the control.

Infection with SARS-CoV-2, B.1 strain, of cells transfected with scFvs anti-N protein
Vero TMPRSS-2 cells plated in d10 dishes and transfected with scFvs were subsequently infected with SARS-CoV-2 B.1 strain at multiplicity of infection (MOI) 0.1 to assess the ability of cells to neutralise the virus.Briefly, the culture medium was removed and cells were inoculated with the virus diluted in a fresh medium supplemented with 5% of FBS.Cells were incubated at 37°C for 3 h.After inoculum, cells were washed with PBS1x and then fresh culture medium with 5% of FBS was added.48 h post infection, supernatants of infected cells were collected and titrated both by limited dilution as described above and by Real Time PCR.

Neutralisation assay
Selected recombinant anti-N scFvs (#5, 24, 77, 255, 261, 336, 291) and a non-related scFv (#58F) were tested individually or in various combination (#5,24; #77,255; #261,336; #291, 255; #291,336; #261,77; #5,77,24,255; #5,261,336,291) in quadruplicate in a neutralisation assay using 10-fold dilutions from 2000 ng/ml up to 0.2 ng/ml and were compared with the control serum 1711, obtained from a convalescent COVID patient, and with the anti-S monoclonal antibodies (MAbs) Bamlanivimab, Etesevimab, and Sotrovimab 3 , obtained from clinical preparations and diluted in physiological solution to therapeutic concentrations.The control serum 1711 was selected amongst 2,000 sera tested for anti-SARS-CoV-2 activity and represented a high titer serum.The serum sample was tested in quadruplicate using 2-fold dilutions from 1:40 to 1:5,120; the MAbs were tested in quadruplicate using 2-fold dilutions from 8 µg/ml.The assay was performed by using an aliquot of B.1 viral strain from titrated stocks.Each aliquot was used only once.Tests were performed in 96well plates containing 100 TCID50/50 µl viral preparation per well that were incubated with 50 µl of scFvs samples or controls diluted in a cell culture medium.This suspension was incubated at 37°C for 1 hour and then supplemented with 10,000 cells/100μ/well of Vero-TMPRSS2 cell lines.Plates were incubated for three days and then examined for cytopathic effect.A control plate was used for viral strain to verify the input titer.

Western blot of VERO-TMPRSS2 cells transfected with scFvs anti-N protein
3x10 5 Vero-TMPRSS2 cells were plated in a 6-well plate and transfected the next day with the scFvs cloned in the pEF1α-IRES-ZsGreen1 plasmid using Lipofectamine LTX (Invitrogen, Thermo Fisher, Italy) according to manufacturer's protocol.Cells were collected 48h after transfection and lysed in an ice-cold RIPA buffer containing protease and phosphatase inhibitor cocktails (Sigma-Aldrich).
Total protein extract was obtained by O/N incubation at 4 °C followed by centrifugation at 12000 rpm for 15 min at 4 °C.Proteins were quantified by Bradford assay (Thermo Fisher Scientific).30 μg of protein lysate were mixed with 5X Laemmli Buffer and denatured at 70 °C for 10 min.Proteins were separated by SDS-PAGE (Biorad) and electro-blotted onto nitrocellulose membranes (Amersham).

Real time PCR of SARS-CoV-2
SARS-CoV-2 RNA relative amounts detected as a cycle threshold (Ct) value for each experimental condition were compared, with a mean Ct value determined for the positive infection control.The viral RNA was purified from 100 μL of cell-free culture supernatant for each condition, using the QIAamp Viral RNA Mini Kit (Qiagen).The purified RNA was then used to synthesize first-strand complementary DNA, using the SuperScript First-Strand Synthesis System for RT-PCR (Thermo Fisher Scientific).To detect the complementary DNA, real-time PCR was performed using the SYBR Green dye-based PCR amplification and detection method.The SYBR Green PCR Master Mix (Thermo Fisher Scientific) was used, with the forward primer N SARS-CoV-2 fwd: TTACAAACATTGGCCGCAAA, and the reverse primer N SARS-CoV-2 rev: GCGCGACATTCCGAAGAA.The PCR conditions were: 95°C for 2 min, 45 cycles of 95°C for 20 s, annealing at 55°C for 20 s and elongation at 72°C for 30 s, followed by a final elongation at 72°C for 10 min.RT-PCR was performed using the ABI-PRISM 7900HT Fast Real Time instrument (Applied Biosystems) and optical-grade 96-well plates.Samples were run in duplicate, with a total volume of Suppl.Fig S1.Analysis of clonotypes overlap between a human naive IgM scFv library and the 6 IgM libraries from Covid-19 recovered patients.Clonotypes were identified using the CDR3 amino acid sequence of the VH and VL Suppl.Fig S2.Overlap occurrences of clonotypes between the VH and VL of the IgM and IgG/IgA libraries from Covid-19 recovered patients.Top panels: Clonotypes occurrences in the VH (right panel) and in the VL clonotypes of the IgM libraries.Bottom panels: Clonotypes occurrences in the VH (right panel) and in the VL clonotypes of the IgG/IgA libraries.
. 8. Size exclusion chromatography profiles of recombinant scFvs.The quaternary structure of recombinant scFvs, and in particular their property of being predominantly in the monomeric form, was tested by SEC analysis.ScFvs #24 and #336 exhibited a good yield and purity, with the monomeric form eluting around 15ml in accordance to the expected MW of 29.5 KDa.On the other hand, ScFvs #5, #93, #255, and #291 showed a high tendency to oligomerize, with almost all protein eluting at the column void volume.ScFvs #93 preparation was divided into fractions A, B: see Supplementary Fig. S23 and S24 for details.
Suppl.Fig S9.Reference frame of the N protein loaded on the Dot Blot membrane.ProNGF represents the negative control.Suppl.Fig S10.Representative Dot Blots carried out on the scFv indicated on the top of the image.(The red arrows indicate binding-positive spots).As shown in Suppl.Fig S10, scFvs 24, 255, 5 and 77 were able to recognize the two higher concentrations of the N protein, scFvs 261 and 336 bound N protein only at the highest concentration, while scFv 291 gave no signal, under the tested conditions.Suppl.Fig. S11.Lack of neutralising activity by anti-N scFvs as recombinant protein (A) and as intracellularly expressed (B, C).A) Comparison of activity of scFv anti-N protein, control serum and anti spike MAbs used in clinics 3 .All purified recombinant scFvs were used either alone or in combination, at concentrations ranging from 2000 ng/ml up to 0.2 ng/ml and compared with a positive-control serum obtained from a convalescent COVID-19 patient and with the anti-S MAbs Bamlanivimab, Etesevimab, and Sotrovimab 3 .B). Anti-N scFvs expression in Vero-TMPRSS2 cells.

Fig. 5 B
Fig. 5 B. Co-immunoprecipitation of SARS-CoV 2 viral N protein with anti N scFvs expressed in Vero TMPRSS2 cells.

Fig. 5 C
Fig. 5 C. Co-immunoprecipitation of SARS-CoV 2 viral N protein with anti N scFvs expressed in Vero TMPRSS2 cells.

Fig. 5 D
Fig. 5 D. Co-immunoprecipitation of SARS-CoV 2 viral N protein with anti N scFvs expressed in Vero TMPRSS2 cells.

Fig. 5 E
Fig. 5 E. Co-immunoprecipitation of SARS-CoV 2 viral N protein with anti N scFvs expressed in Vero TMPRSS2 cells.

Fig. 5 F
Fig. 5 F. Co-immunoprecipitation of SARS-CoV 2 viral N protein with anti N scFvs expressed in Vero TMPRSS2 cells.

Suppl. Table S2: NGS libraries sequencing data. For
IgA and IgG ELISA kits® detecting IgA or IgG against a recombinant form of the S1 subunit (Euroimmun, Diagnostica Medica, Italia).In bold are highlighted the 6 patients from which the scFvs libraries (6 IgM and 6 IgG/IgA) were prepared.
Suppl.TableS1: Information of the 40 patients who had recovered from COVID-19 enrolled for biological samples collection of and PBMC isolation.Blood samples were collected by the Virology Unit of the Azienda Ospedaliero Universitaria Pisana (AOUP) and screened for the presence of COVID-19 specific neutralising antibodies measured (see below for description).Anti S antibodies titer as measured using SARS-CoV-2Plates were incubated for three days and then examined for cytopathic effect.A control plate was used for viral strain to verify the input titer.